Many cell stimulators act via cell surface receptors, G proteins, and effector enzymes that transmit the signal. Regulators of G protein signaling (RGS proteins) inactivate the signal by accelerating G protein GTPase activity. Our hypothesis is that many RGS proteins not only regulate but also transmit signals (i.e. act as effectors). Our approach will be to identify signaling events requiring the N-terminal domain of the yeast RGS protein, Sst2 (N-Sst2 domain). There are four specific aims of the proposal: (1) Identify protein mediators of the signal transmitter function of N-Sst2. Microarray analysis has revealed a set of genes induced by N-Sst2, most having a stress response element (STRE) in their promoter region. To identify components of the N-Sst2 signaling pathway, we will compare stress activation in cells that lack or over express known components of the mating- and stress-response pathway, and conduct a genetic screen for additional components that regulate both pathways. (2) Determine the mechanism of N-Sst2 signal transduction. Two-hybrid screening has revealed a number of proteins that bind selectively to N-Sst2. Each binding partner will be deleted or overexpressed to establish those that mediate Sst2 signaling, and to identify those that modulate its G protein-regulatory function. Selected binding partners will be purified and reconstituted to determine their mechanism of action. (3) Identify and characterize the Sst2 protease. Sst2 is proteolytically processed in vivo to yield separate N- and C-terminal fragments. To determine how processing alters Sst2 signaling, we will screen for protease resistant mutants of Sst2 and mutants that lack the Sst2-protease. To determine how processing is regulated, we will screen for mutants that mimic a protease-resistant form of Sst2, and for proteins that interact with the Sst2 protease. (4) Identify mammalian homologues of Sst2 regulators and effectors. There are likely to be mammalian homologues of N-Sst2-binding proteins and the Sst2-protease. Heterologous expression, genetic screening, and bioinformatic strategies will be used to identify factors that confer N-Sst2-like activity on mammalian RGS proteins expressed in yeast, and eventually in mammalian cells.